This invention pertains to a method to detect 2-methylisoborneol using monoclonal antibodies and to a hybridoma cell line that produces such monoclonal antibodies.
Metabolites synthesized by some cyanobacteria, actinomycetes, and fungi are responsible for the earthy/musty or xe2x80x9coff-flavorxe2x80x9d taste in potable water and certain fish. See S. W. Krasner, xe2x80x9cAnalytical methods for the identification and quantification of earthy/musty flavors in drinking water: A review,xe2x80x9d Water Quality Bulletin, vol. 13, pp. 78-83 (1988); and P. B. Johnsen et al., xe2x80x9cPhysiological approaches to the management of off-flavors in farm-raised channel catfish, Ictalurus punctatus,xe2x80x9d in Recent Developments in Catfish Aquaculture, pp. 141-161 (1994). Two metabolites, which are the primary compounds responsible for this xe2x80x9coff-flavor,xe2x80x9d are geosmin (1xcex1, 10xcex2-dimethyl-9xcex1-decalol) and 2-methylisoborneol (1-R-exo-1,2,7,7-tetramethyl-bicyclo-[2.2.1]-heptan-2-ol) (xe2x80x9cMIBxe2x80x9d). These two compounds cause problems for the potable water and aquaculture industries because of the low concentrations of the compounds required to produce the xe2x80x9coff-flavorxe2x80x9d taste and because of the ubiquitous nature of the organisms that produce these metabolites.
The problem with earthy/musty flavors constitutes a significant restriction to the growth of the catfish industry. C. S. Tucker et al., xe2x80x9cEnvironment-related off-flavors in fish,xe2x80x9d in Aquaculture and Water Quality, D. E. Brune and J. R. Tomasso, eds., pp. 133-179 (1991). Catfish cultured in ponds in the southern United States that are deemed unacceptable because of a xe2x80x9cmustyxe2x80x9d flavor can be as high as 80% at any one time. J. F. Martin et al., xe2x80x9cPharmacokinetics and tissue disposition of the off-flavor compound 2-methylisoborneol in the channel catfish (Ictalurus punctatus), Can. J. Fish. Aquat. Sci., vol. 47, pp. 544-547 (1990); J. F. Martin et al., xe2x80x9cMusty odor in chronically off-flavored channel catfish: Isolation of 2-methyleneboranane and 2-methyl-2-bornene,xe2x80x9d J. Agric. Food Chem., vol. 36, pp. 1257-1260 (1988); and J. F. Martin et al., xe2x80x9c2-methylisoborneol implicated as a cause of off-flavor in channel catfish, Ictalurus punctatus (Rafinesque), from commercial culture ponds in Mississippi,xe2x80x9d Aquaculture and Fisheries Management, vol. 19, pp. 151-157 (1988). When xe2x80x9cmustyxe2x80x9d fish cannot be harvested and brought to the market, the fish are held and fed until deemed xe2x80x9con-flavorxe2x80x9d by an experienced human taster employed by the processing plant. However, during this time the fish grow beyond their optimal market size. Fish greater than about an 11 oz fillet are considered undesirable to both the producers and processors because they are more expensive to keep, the fat content of the fillet increases, automatic filleting is more difficult, and fish survival rates decrease as holding times increase.
Additionally, the earthy/musty flavors have been identified as a problem with trout and in potable water. See, for example, M. Yurkowski et al., xe2x80x9cIdentification, analysis, and removal of geosmin from muddy-flavored trout,xe2x80x9d J. Fish. Res. Board Can., vol. 31, pp. 1851-1858 (1974); G. Izaquirre et al., xe2x80x9cGeosmin and 2-methylisoborneol from cyanobacteria in three water supply systems,xe2x80x9d Applied and Environmental Microbiology, vol. 43, pp. 708-714 (1981); and Aoyama, xe2x80x9cStudies on the earthy-musty odors in natural water (IV). Mechanism of earthy-musty odor production of actinomycetes,xe2x80x9d Journal of Applied Bacteriology, vol. 68, pp. 405-410 (1990).
Methylisoborneol (xe2x80x9cMIBxe2x80x9d) is a small terpenoid compound synthesized by cyanobacteria and actinomycetes and is illustrated below. It is fat-soluble and not easily volatilized.

MIB is believed to be taken up by fish through their gills and stored in fatty tissues. Fish with higher fat reserves are more prone to a xe2x80x9cmustyxe2x80x9d flavor. Research related to quality control of catfish flavor has focused on the pond management by studying factors that influence the growth of algae or fungi populations, such as water quality, pH, oxygen levels, soil quality, fish density, feed quality and application rates, and water temperature. See, e.g., H. G. Peterson et al, Physiological toxicity, cell membrane damage, and the release of dissolved organic carbon and geosmin by Aphanizomenon flos-aquae after exposure to water treatment chemicals,xe2x80x9d Wat. Res. Vol. 29, pp. 1515-1523 (1995); C. P. Dionigi et al., xe2x80x9cCopper-containing aquatic herbicides increase geosmin biosynthesis by Streptomyces tendae and Penicillium expansum,xe2x80x9d Weed Science, vol. 43, pp. 196-200 (1995); and R. Velzeboer et al., xe2x80x9cRelease of geosmin by Anabaena circinalis following treatment with aluminum sulphate,xe2x80x9d Wat. Sci. Tech., vol. 31, pp. 187-194 (1995). Although many approaches to eliminate algal growth have been tried, some algal growth is beneficial to maintain an adequate oxygen content in the water during daylight hours, especially when the temperature of the water is high. Because of this interaction between algae and oxygen and because of the large numbers of factors that affect both algal and fungal growth, controlling the xe2x80x9coff-flavorxe2x80x9d by pond management to control the algal density has proven difficult.
Another problem compounding the problem of xe2x80x9cmusty flavorxe2x80x9d in aquaculture products is the difficulty in easily measuring the metabolites responsible. The xe2x80x9cmusty flavorxe2x80x9d is caused by metabolites that are present at extremely low concentrations. The two methods used currently to detect both geosmin and MIB are human tasters and gas chromatography (xe2x80x9cGCxe2x80x9d). The human gustatory threshold of geosmin or MIB is approximately 10 to 30 ng/L in pure water. See, e.g., J. A. Maga, xe2x80x9cMusty/earthy aromas,xe2x80x9d Food Reviews International, vol. 3, pp. 269-284 (1987); P. -E. Persson, xe2x80x9cSensory properties and analysis of two muddy odor compounds, geosmin and 2-methylisoborneol, in water and fish,xe2x80x9d Water Research, vol. 14, pp. 1113-1118 (1980); and S. F. Wood et al., xe2x80x9c2-Methylisoborneol, improved synthesis and a quantitative gas chromatographic method for trace concentrations producing odor in water,xe2x80x9d J. Chromatography, vol. 132, pp. 405-420 (1977). Human sensory evaluation is the most sensitive method of detection currently available, but this method is subjective with a large degree of variation between tasters and even between tests by the same tester. Despite this problem, necessity has dictated the use of human flavor-tasters at catfish processing plants who have the authority to accept or reject catfish prior to harvest or delivery.
Gas chromatography (xe2x80x9cGCxe2x80x9d) has been used to measure geosmin and MIB in analytical laboratories, but the use of this method by industry is limited. See, e.g., H. P. Dupuy et al., xe2x80x9cAnalysis for trace amounts of geosmin in water and fish,xe2x80x9d JAOCS, vol. 63, pp. 905-908 (1986). Analysis by GC requires that all hydrophilic species be completely removed from the sample. Thus, fish samples must be extracted and prepared using multi-step procedures that give low recovery rates and poor reproducibility. Recovery rates for known controls can be as low as 40 to 50%. More importantly, GC analysis requires skill and training, is costly, and is not adaptable to mass screening.
There is an unfilled need for a method to measure MIB that is more reliable and facile than either the sensory or gas chromatographic analyses currently available.
An immunoassay method to measure MIB has been tried. Polyclonal antibodies (PAb) were produced by binding compounds similar in structure to geosmin and MIB (argosmin and camphor, respectively) to a carrier protein to create a conjugate. The conjugate was then used to elicit an immune response and subsequent production of polyclonal antibodies. See S. -Y. Chung et al., xe2x80x9cDevelopment of an ELISA using polyclonal antibodies specific for 2-methylisoborneol,xe2x80x9d J. Agric. Food Chem., vol. 38, pp. 410-415 (1990); Chung et al., xe2x80x9cDevelopment of an enzyme-linked immunosorbent assay for geosmin,xe2x80x9d J. Agric. Food Chem., vol. 39, pp. 764-769 (1991); and Chung et al., xe2x80x9cAttempts to improve the sensitivity of an enzyme-linked immunosorbent assay for 2-methylisoborneol,xe2x80x9d Water Sci. Technol., vol. 25, pp. 89-95 (1992). The polyclonal antibodies were unacceptable because of high non-specific binding and poor sensitivity (only detected down to 1 xcexcg/ml), and because of the generation of false-positives from the presence of non-odiferous, structurally-related metabolites (e.g. argosmin, 2-methyleneborane and 2-methyl-2-borene). This poor sensitivity meant the PAbs were not useful to measure the low levels of MIB that causes xe2x80x9coff-flavorxe2x80x9d in foods. The detection limit using these PAbs was at least three orders of magnitude above the human sensory threshold and thus too high to be of practical use.
Monoclonal antibodies to geosmin analogs and geosmin derivatives bound to bovine serum albumin have been reported. See, B. L. Middlebrooks et al., xe2x80x9cA method for the production of monoclonal antibodies against geosmin (trans-1,10-dimethyl-trans-(9)-decalol),xe2x80x9d Abstract of a Paper presented at the Annual Meeting of the American Society for Microbiology, Abstract No. Q-133, Atlanta, Ga., 1987. There was no indication of the sensitivity of the MAbs.
Monoclonal antibodies have been reported to MIB, without disclosing the hybridoma cell-line. See Park et al., xe2x80x9cDevelopment of monoclonal antibody and enzyme immunoassay for 2-methylisoborneol,xe2x80x9d Abstract of a Paper presented at 4th International Conference on Toxic Cyanobacteria, Sep. 27-Oct. 1, 1998, Beaufort, N.C.
Areas in which a sensitive assay for MIB would be useful include measuring the quality or safety of potable water supplies, pond water, water filtering devices, aquacultural or fishery products (e.g., catfish, trout, shrimp, oysters, and clams), and agricultural products (e.g., hydroponic water supplies, and aquatic plants). Additionally, such an assay could monitor indoor air quality (e.g., fungal growth in air ducts), indicate a fungal infection in blood, and indicate fungal growth in grain storage containers.
We have produced a hybridoma cell line (F6b4G7b4, ATCC No. PTA-911) that produces monoclonal antibodies (MAb) that bind strongly to MIB. The MAbs were produced using a structurally related molecule, borneol, whose structure was minimally changed when complexed to the protein carrier. These MAbs detected MIB at approximately 0.01 to 0.1 ppb or lower when used in ELISA assays. Because these antibodies were produced as MAbs, their affinity characteristics will remain constant. Using these MAbs, MIB can be easily detected at low levels using immunological techniques that are adapted to fast and easy field assays to test a large number of samples.